Toll-Like Receptors (TLRs) are conserved membrane pattern recognition receptors (PRRs) of innate immunity responsible for clearing microbial pathogens. TLRs are expressed in many immune and non-immune cells and detect pattern recognition motifs that recognize microbial products, namely, the pathogen associated molecular patterns (PAMPs, ex: nucleic acids, lipoprotein and polysaccharides) (Kawai et. al., 2011, Immunity, 34(5):637-50). In addition to PAMPs, several TLRs also recognize endogenous ligands resulting from cellular insult due to inflammatory response and cell-death, called damage associated molecular patterns (DAMPs) (Abdelsadik et. al, 2011, Human Immunology, 72, 1188-1193). Till date, ten functional TLRs (TLR1-10) have been identified in human, of which TLR1, TLR2, TLR4, TLR5 and TLR6, that recognize lipoproteins and lipopolysaccharides are expressed on the plasma membrane and TLR3, TLR7, TLR8 and TLR9 that recognize nucleic acids are expressed in endosomal compartments. While TLR7 and TLR8 both recognize ssRNA, TLR9 recognizes CpG-rich hypomethylated DNA (Table 1).
TABLE 1Summary of Function and Expression of TLR7 and 9TLRCellular ExpressionPAMPs (Microbial)DAMPs (Host)TLR7B-cells, pDCsssRNAssRNA,Antiphospholipidantibodies,cardiac myosinTLR9B-cells, pDCs, GICpG DNADNA, DNAepithelial,containingKeratinocytesimmunecomplexes, IgG-chromatincomplexes
Engagement of a cognate ligand to TLRs induces conformational changes allowing formation of homo- or heteromeric interactions within TLRs and recruitment of adaptor proteins such as MyD88, TIRAP, TRIF, and TRAM. TLR7 and TLR9 are localized mainly to ER in the steady state but traffic to the endo/lysosomal compartment with the help of UNC93b1 during activation (Kawai T and Akira S, 2007, Sem Immunol, 19, 24). TLR7 and TLR9 activate NF-κB and IRF7 via MyD88 to induce pro-inflammatory cytokines (TNFα, IL-1β, IL-6) and type I interferons (IFNα and IFNβ) respectively. The activation of NF-κB during TLR7 and TLR9 signaling is initiated from the endosomes whereas IRF7 activation is initiated from the lysosome-related organelle (LRO) after TLR7 and TLR9 are transported from the endosome to this vesicle. MyD88-dependent IRF7 activation in pDCs is mediated by activation of IRAK1, TRAF6, TRAF3 and IKKα. In conventional DCs and macrophages, TLR7 and TLR9 induce inflammatory responses by activating NF-κB via MyD88 but fail to activate IRF7 [Kawai et. al., 2011, Immunity, 34(5):637-50].
TLR7/9 activation plays a major role in the inter-phase of innate and adaptive immunity. They not only activate inflammatory cytokines, up-regulate MHC molecules and co-stimulatory signals in antigen-presentation (innate immune response) but also prime and amplify T-, and B-cell effectors function (adaptive immune response) [Hannessy et. al., 2010, Nat Rev Drug Discov., 9(4):293-307; Koegh et. al., 2011, Trends Pharmacol Sci, 32(7):435-42].
There is increasing evidence of role of TLR-7 and 9 in the pathogenesis of various autoimmune diseases including rheumatoid arthritis (RA). Human synovial tissue from RA patients showed the expression of TLR7 and 9 (along with TLR-2/3/4). The expression of TLR7 was significantly up-regulated in RA synovial fibroblasts (RASFs) compared with healthy controls or patients with non-inflammatory arthritis. Stimulation of cultured RASFs with TLR7 ligands resulted in significant up-regulation of chemokines, cytokines, metalloproteinases and type I IFNs. [Roelofs et. al., 2005, Arthritis Rheum. 52(8):2313-22].
Recent studies have shown that the development and progression of Systemic Lupus Erythematosus (SLE) are driven by the over-expression of TLR-7, 8 and 9 within B-cells and pDCs [Komatsuda et. al., 2008, Clin Exp Immunol.; 152 (3):482-7; Migita et. al., 2007, J Rheumatol., 34 (3): 493-500]. A knockout of TLR7 in a spontaneous murine model of SLE (MRL−/Mplpr/lpr), showed decreased anti-RNA antibodies, diminished spelinic immune activation and suppressed the development of nephritis [Nickerson et. al., 2010, J Immunol., 15, 184 (4):1840-8; Christensen et. al., 2006, Immunity, 25 (3): 417-28], validating the target in the pathogenesis of this disease.
Antagonists of these nucleic acid-recognizing TLRs have primarily been oligonucleotide-based molecules. IMO-3100, an oligonucleotide based antagonist of TLR7 and TLR9 has demonstrated clinical activity in psoriasis patients. This antagonist has also shown encouraging results in mouse models of lupus, collagen induced arthritis and psoriasis. IMO-8400, a TLR-7/8/9 antagonist has been shown to suppress the production of autoimmune antibodies, improve kidney histopathology and decrease blood urea nitrogen and proteinuria in lupus prone mice.
The small molecule anti-malarial drugs like chloroquine, hydroxychloroquine and quinacrine, that are also known to show TLR-7/8/9 antagonism, have been used since the 1950s to treat immune-mediated inflammatory disorders (IMID) such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). However, side effects associated with these drugs and also the suboptimal efficacy has limited the use of these anti-malarials in IMID.
There is literature prior art disclosing small molecule TLR-7/8/9, TLR-7/9 and TLR9 antagonists. Coley Pharmaceuticals has disclosed series of 4-aminoquinoline derivatives as inhibitors of immune stimulation involving at least one of TLR9, TLR8, TLR7 and TLR3 (U.S. Pat. No. 7,410,975). Eisai R & D has disclosed benzoxazole compounds as antimalarials, claiming the compounds being antagonists of TLR9 (WO 2010/036908). In a patent application (WO 2011/115183), Dainippon Pharma has disclosed monocyclic pyrimidine derivatives that are effective in the prophylaxis and/or treatment of disorders associated with signal transmission mediated by Toll-like receptors (TLR). The biological data disclosed in the application substantiate the claimed compounds as TLR9 antagonists. In a very recent patent application by Janus Biotherapeutics (WO 2013/052550), imidazoloquinoline-based compounds have been described as immune system modulators. Two more patent applications from Janus Biotherapeutics (WO 2012/167046 and WO 2012/167053) have described pyrazinopyrimidines and imidazolopyrimidines as immune system modulators. In all the three patent applications, biological data reveal that the claimed compounds demonstrate TLR9 antagonism.
Despite several discoveries in this area, there are no safe and efficacious orally administered small molecule TLR-7/8/9 antagonists available in the market. CPG-52364 from Coley Pharma and Pfizer has been discontinued from clinical trials in 2008 for unknown reason. Therefore, there is a strong need for a novel small molecule TLR-7/8/9 antagonist that will be orally available and will have potential clinical utility. These compounds will have medical application in the disease area of inflammation, autoimmune disorder and cell proliferation, rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic lupus erythematosus, Sjögren's Syndrome, multiple sclerosis, inflammatory bowel disease, allergic diseases, infectious diseases affecting immune system, asthma, type 1 diabetes, myasthenia gravis, hematopoetic disfunction, B-cell malignancies, transplant rejection and graft-versus-host disease.
Patent Document 1 discloses a compound represented by the formula:
wherein each symbol is as defined in the specification, as a Bruton's tyrosine kinase inhibitor.